Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan.
Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan.
Enzyme Microb Technol. 2019 Feb;121:17-22. doi: 10.1016/j.enzmictec.2018.10.014. Epub 2018 Oct 31.
Various recombinant proteins can be produced by the yeast Saccharomyces cerevisiae cell factories; therefore, efficient recombinant protein production techniques are desirable. In this study, to establish an efficient recombinant protein production technique in S. cerevisiae, the secretory production of recombinant protein endoglucanase II (TrEG) was tested. We developed 2 novel methods for TrEG production via clustered regularly interspaced short palindromic repeat (CRISPR) -δ-integration as well as multiple promoter shuffling, which involved the pre-breakdown of the δ-sequence by the CRISPR system and subsequent δ-integration as well as the conjugation of TrEG with various promoters and subsequent δ-integration, respectively. Moreover, simultaneous use of the CRISPR-δ-integration and multiple promoter shuffling methods was also examined. The CRISPR-δ-integration method was effective for improvement of the integrated TrEG copy number and its activity, and the multiple promoter shuffling method was also beneficial for enhancing the transcriptional level of TrEG and its activity. Furthermore, simultaneous use of CRISPR-δ-integration and multiple promoter shuffling methods was the most useful. The carboxymethyl cellulase activity of the TrEG expressing transformant YPH499/24CP constructed by the method reached 559 U/L, and it was 17.3-fold higher than that of the transformant constructed by the conventional YEp type vector. Overall, the simultaneous use of CRISPR-δ-integration and multiple promoter shuffling can be useful and easily applied for recombinant protein production.
各种重组蛋白可由酵母酿酒酵母细胞工厂生产；因此，需要高效的重组蛋白生产技术。在本研究中，为了在酿酒酵母中建立一种高效的重组蛋白生产技术，测试了重组内切葡聚糖酶 II（TrEG）的分泌生产。我们通过成簇规律间隔短回文重复序列（CRISPR）-δ-整合以及多个启动子改组开发了 2 种新型 TrEG 生产方法，分别涉及 CRISPR 系统对内切酶 δ-序列的预破坏和随后的 δ-整合以及 TrEG 与各种启动子的连接和随后的 δ-整合。此外，还检查了同时使用 CRISPR-δ-整合和多个启动子改组的方法。CRISPR-δ-整合方法有效地提高了整合的 TrEG 拷贝数和其活性，而多个启动子改组方法也有利于提高 TrEG 的转录水平及其活性。此外，同时使用 CRISPR-δ-整合和多个启动子改组方法最为有用。通过该方法构建的表达 TrEG 的转化子 YPH499/24CP 的羧甲基纤维素酶活性达到 559 U/L，比通过常规 YEp 型载体构建的转化子高 17.3 倍。总的来说，同时使用 CRISPR-δ-整合和多个启动子改组可以是有用的，并且易于应用于重组蛋白生产。
